Gene fusions with .beta.-galactosidase (.beta.-gal) have been used to monitor gene expression in E. coli (1, 2, 3, 32). Such fusions have been used with limited success in the study of secretory proteins. .beta.-gal fusions of such proteins can be lethal to the host and in cases of nonlethality the .beta.-gal is active whether it remains cytoplasmic or is secreted to the periplasmic space (4).
Alkaline phosphatase (AP) has been shown to have enzymatic activity when it is secreted to the periplasm (5). This activity can be visually estimated by the intensity of the blue color of colonies grown on agar plates containing 5-Bromo-4-chloro-3-indolyl phosphate (X-P) (6). Wright has shown examples where gene fusions at the amino terminus of AP preserved enzymatic activity and that the first 33 amino acids of the amino terminus of AP were not required for activity (7). Additionally, AP can be secreted to the periplasm at a high level in E. coli when carried on a multicopy plasmid (6). In this connection see European Patent Application 77,569.
Methods are known for the secretion of heterologous proteins into bacterial periplasmic space (33, 34). However, no convenient methods are available to the art which would facilitate the screening of large numbers of transformants in order to identify host cells or vector constructions that secrete high levels of desired protein. Instead, each transformant has been cultured individually, periplasmic product recovered and the efficacy of the host-vector system evaluated by product assays.
Accordingly, it is an object of this invention to provide a method for screening transformants to identify those having the ability to secrete maximal levels of heterologous protein.
It is a further object herein to provide a method for rapidly and economically identifying mutants within the coding and noncoding regions of the DNA to be expressed which enhance the expression and/or secretion of the desired heterologous protein, as well as mutants in the host cell which exert a similar effect.
These and other objects will be apparent to the ordinary artisan from consideration of the specification as a whole.